This PPG has documented the existence of a paracrine signaling loop between tumor cells and tumor- associated macrophages (TAMs), in which reciprocal production of EGF and GSF-1 interact with cognate receptors in each cell type to promote invasion and metastasis. Recently, we have uncovered a role for G- protein coupled receptor (GPCR) activation of PI S-kinase (PISK) in the macrophage-tumor cell paracrine loop. We have generated extensive preliminary data implicating GPCR signaling and GPCR-dependent PISKs in both arms of the paracrine loop (macrophage to tumor cell and tumor cell to macrophage). The present proposal will examine the role of GPCR signaling in macrophage-tumor cell interactions. Aim 1 takes advantage of novel mutants of the two GPCR-regulated PISKs, pHOy and p110(3, which are specifically defective for activation by GPCRs. We will disrupt GPCR-mediated signaling in tumor cells and macrophages by replacing endogenous pHOp/y with these mutants, and then evaluate paracrine signaling between the cells using assays developed by this PPG: SD invasion, intravasation and extravasation in vitro, and Invasion, experimental metastasis, and in vivo motility assays using intravital Imaging in animals. Aim 2 focuses on the PlSK-dependent phosphorylation of tumor cell Nonmuscle Myosin Heavy Chain-llA (NMHC- llA) S194S, which promotes NMHC-llA disassembly and enhances macrophage-dependent tumor cell invasion. We will directly test whether S194S phosphorylation in tumor cells is regulated by paracrine signals from macrophages. We will express phosphorylation site mutants of NMHC-llA in tumor cells and macrophages, and measure effects on invasion, Intravasation and extravasation in vitro and in vivo. We will use breast tumor reverse phase protein arrays (RPPAs) to determine If increased phospho-NMHC-IIA correlates with metastatic progression in cancer patients, and we will use a high throughput siRNA kinase screen to identify the kinases that directly regulate NMHC-llA phosphorylation in tumor cells. Finally, Aim 3 Is based on data suggesting a role for PISKs and NMHC-llA in the reciprocal stimulation of chemokine secretion by macrophages and tumor cells. We will test whether the expression of mutant PISKs and NMHC- llA alters the secretion of chemokinetic factors by each cell, as well as the related process of MMP trafficking to Invadopodia in tumor cells, and examine the mechanism for these secretory phenotypes. Overall, this proposal will provide important new insights in GPCR regulation of macrophage-tumor cell interactions. RELEVANCE (See instructions): Therapeutic approaches targeting tumor metastasis are a critical priority in current cancer research. Previous work from this program project has shown that interactions between tumor cells and surrounding tissue profoundly influence tumor cell invasion and metastatic spread. This proposal provides new insights into the molecular basis for these interactions, and may lead to the identification of novel therapeutic targets.